Viruses represent major disease transmitting agents carried by human excreta and animal manure. Understanding virus inactivation is therefore essential in preventing microbial spread due to inadequate treatment of these materials. Here, we investigated the inactivation kinetics of the single-stranded (ss) RNA phage MS2, DNA phages T4 and ΦX174, and the double-stranded DNA human adenovirus in stored human urine, sludge, and animal manure, at temperatures and pH values typical of storage under naturally occurring conditions or mesophilic anaerobic digestion (< 40 °C). The ssRNA phage MS2 was most readily inactivated in all samples compared to the other viruses tested. This is consistent with previous findings in well-controlled buffer solutions of similar composition, where inactivation was found to be governed by bases (NH3, carbonate, hydroxide) that catalyze the transesterification and cleavage of the ssRNA. Correspondingly, MS2 inactivation kinetics in real matrices could be adequately modelled by only taking into account the effects of temperature, pH, carbonate and ammonia on the integrity of ssRNA. DNA viruses were more persistent compared to MS2; however, inactivation in selected sludge and manure samples proceeded at faster rates compared to well-controlled buffer solutions of similar composition. This indicates a contribution of microbial or enzymatic activity to inactivation of DNA viruses. Overall, this study identifies the most important factors contributing to inactivation of viruses in human excreta and manure, and highlights the differences in inactivation kinetics and mechanisms between ssRNA and DNA viruses.